Aire is a transcriptional regulator that controls immunological tolerance by driving the expression of a large repertoire of gene transcripts encoding peripheral-tissue self-antigens in thymic medullary epithelial cells (MECs). Thereby, it promotes both the negative selection of effector T cells capable of recognizing these self- antigens and positive selection of regulatory T cells that can rein in the effectors. Mutations in the AIRE gene underlie a rare multi-organ autoimmune disease called APECED or APS-1, and abnormalities in the Aire pathway, reflecting polymorphisms in either Aire target-loci or in the AIRE gene itself, have also been implicated in certain of the more common autoimmune disorders, notably type-1 diabetes, myasthenia gravis, vitiligo and pernicious anemia. The studies proposed herein issue from our surprising finding that Aire's impact on immunological tolerance has a strikingly narrow temporal window: its expression during the perinatal period is necessary and sufficient to avoid the multi-organ autoimmune disease typical of Aire-knockout mice. Hence, this project's overall goal is to understand why Aire must be expressed in perinatal mice to guard against autoimmunity, but is dispensable in adults. Its Specific Aims are to: 1. evaluate the role of DM and DO in the selection of Foxp3+CD4+ Treg cells; 2. explore the fate of perinatally generated Tregs; and 3. compare perinatal and adult CD4+ Teff cells and Aire's impact thereon. Addressing these Aims will entail the generation of new lines of multi-engineered mice, tailored to highlight the impact of designated molecules (e.g. Aire, DM) or cell-types (e.g. ?:? T cells); as well as state-of-the-art approaches to the genomic analysis of ultra small immunocyte populations. Results from these studies will not only elucidate mechanisms underlying the temporal window of Aire's impact on immunological tolerance but, more generally, should yield new insights into the molecular and cellular underpinnings of neonatal tolerance, a phenomenon that has fascinated immunologists for decades. Autoimmune diseases afflict 7-10% of Americans, and are rising in incidence. It is imperative to learn how to re-establish immunological tolerance in individuals with autoimmune disorders - understanding tolerance mechanisms will undoubtedly advance this goal.